Numerous developmental processes are regulated by signaling cascades that alter protein phosphotyrosine levels. Many extracellular cues are linked to cellular responses via transmembrane receptor protein-tyrosine kinases (PTKs) and phosphatases (PTPs). In the nervous system, transmembrane kinases and phosphatases are required for neuronal differentiation and survival, neurite extension, the directed growth of the neuronal growth cone, and the fasciculation of nerve bundles (Snider, Cell 77:627-638 (1994); Callahan et al., Nature 376;171-174 (1995); Tessier-Lavigne, Cell 82:345-348 (1995); Desai et al., Cell 84:599-609 (1996); Krueger et al., Cell 84:611-622 (1996)). These transmembrane receptors are directly regulated by specific ligands. Cytoplasmic PTKs are also involved in the development of the nervous system, although the ligands which induce their activation are less well understood (Gertler et al., Cell 58:103-113 (1989); Grant et al., Science 258:1903-1910 (1992); Umemori et al., Nature 367:572-576 (1994)). There is growing evidence that these kinases are regulated in pathways responding to components of the extracellular milieu and may function to regulate axonal growth downstream of receptors that lack intrinsic kinase activity (Bixby & Harris, Ann. Rev. Cell. Biol. 7:117-159 (1991)).
The non-receptor PTK Src is highly expressed in the developing mammalian nervous system (Maness et al., Adv. Exp. Med. & Biol. 265:117-125, (1990); Maness, Dev. Neurosci. 14:257-270 (1992)). During neurogenesis Src kinase activity increases and Src becomes concentrated in growth cones of neurons. Growth cones migrate by extending actin-rich filopodia and lamellipodia, and tyrosine phosphorylation is important for the formation of these actin structures (Wu & Goldberg, J. Cell Biol. 123:653-664 1993; Goldberg & Wu, J. Neurobiol. 27:553-560 (1995)). Neurons cultured from mice that lack Src extend neurites less well than wild-type neurons when plated on surfaces coated with the neural cell adhesion molecule L1 (Ignelzi et al., Neuron 12:873-884 (1994)). This defect is specific, since neurons lacking the Src relatives Fyn or Yes extend neurites normally (Beggs et al., J. Cell Biol. 127:825-833 (1994)). Moreover, neurons from mice lacking Fyn extend only short neurites on NCAM-140 but extend long neurites on L1 (Beggs et al., ibid.). Src and Yes are not needed for neurite extension on NCAM-140. These specific defects point to the existence of adhesion-stimulated, Src- and Fyn-dependent, regulatory processes required for neurite extension. Signals from neurotrophin receptor PTKs, such as TrkA, may also be relayed through Src. Nerve growth factor-(NGF) induced neurite extension is Src dependent in PC12 pheochromocytoma cells (Kremer et al., J. Cell Biol. 115:809-819 (1991); Vaillancourt et al., Mol. Cell. Biol. 15:3644-3653 (1995)).
The non-receptor tyrosine kinase, Abl, participates in nervous system development in Drosophila. The Drosophila Abl (dAbl) protein is found in many cell types in the developing embryo, but expression is highest in the cell bodies and axons of neurons in the developing central nervous system (CNS) (Gertler et al., ibid. (1989); Bennett & Hoffmann, Devel. 116:953-966 (1992)). Flies lacking the dAbl gene develop past metamorphosis but die as adults before or soon after eclosion (Henkemeyer et al., Cell 51:821-828 (1987)). Five genes were identified in screens for dominant second site mutations that exacerbate the dabl− phenotype and have been dubbed HDA (haploinsufficient, dependent upon dAbl) genes (Gertler et al., ibid., (1989); Hill et al., Genetics 141:595-606 (1995)). When heterozygous for a mutation in a HDA gene, dAbl− but not dAbl+ embryos die as embryos, with a characteristic terminal phenotype. The neurons of the CNS are present in normal number and extend axons, but gaps are apparent in the commissural and longitudinal axon bundles (Gertler et al., ibid. (1989), Gertler et al., Genes Dev. 7:441-453 (1993); Hill et al., ibid. (1995)). Three of the HDA genes, disabled (dab), prospero, and fax have been cloned and have distinct properties (Vaessin et al., Cell 67:941-953 (1991); Gertler et al., ibid. (1993); Hill et al., ibid. (1995)). Homozygous mutations in the HDA genes dab and fax in a dAbl mutant background results in almost complete loss of CNS axonal tracts. The dab and fax genes also show dosage sensitive interactions with each other (Gertler, “Genetic Modifiers of the Drosophila abl mutant phenotype,” Ph.D. Dissertation, University of Wisconsin-Madison (1992); Hill et al., ibid. (1995)) as well as with dAbl and therefore may have related functions.
The Drosophila dab gene encodes a 2412 residue protein (Dab) that co-localizes with dAbl to the cell bodies and axons of embryonic CNS neurons (Gertler et al., ibid. (1993)). In Drosophila Dab is essential for normal CNS development, even in the presence of dAbl. Dab is tyrosine phosphorylated in insect cells and, given the co-localization with dAbl in the CNS, it has been suggested that Dab may be a physiological substrate of dAbl (Gertler et al., ibid. (1993)). However, the role of tyrosine phosphorylation in regulating Dab function, and the identities of the PTKs that phosphorylate Dab, remain unclear. The kinase activity of dAbl is dispensable for normal embryonic development, unless the levels of Dab or other HDA gene products are reduced by heterozygous mutations (Henkemeyer et al., Cell 63:949-960 (1990)). Using a temperature-sensitive mutant, dAbl kinase activity was shown to be required in dab heterozygotes after the time of cell fate specification and during the time of axonogenesis in the embryonic CNS (Henkemeyer et al., ibid. (1990)). Despite the loss of nerve bundles in the CNS, the total number of neurons is unaffected (Gertler et al., Science 248:857-860 (1990)). These results suggest that dAbl has kinase dependent and independent roles in development. Other PTKs that are expressed in the fly CNS, such as Drosophila Src(dSrc) (Simon et al., Cell 42:831-840 (1985)), may substitute for the dAbl kinase requirement in CNS development, provided the levels of Dab are normal.